The optical measurement of reactions occurring at the single molecule level provides the ability to probe molecular interactions and molecular mechanisms that are not readily accessible when measured on ensembles of molecules. In particular, observing a single polymerase enzyme while it sequentially adds nucleotides to a growing strand provides an extremely effective method for performing nucleic acid sequencing.
Optical confinements such as zero-mode waveguides provide for the observation of single molecules by producing a small optical observation volume, allowing for detection of signal from the reaction of a single molecule within the volume without the background signal which would be present in a larger observation volume. Substrates having thousands of optical confinements can be produced commercially to allow the monitoring of thousands of single molecules simultaneously.
Arrays of optical confinements having polymerase enzymes immobilized within them provide for the parallel sequencing of thousands of template nucleic acids at one time.
While arrays of optical confinements can be used for nucleic acid sequencing applications, the detection of single molecules provides an inherently small signal, which must be observed in the presence of background signals. There is a need for systems and methods that can more effectively identify and/or remove observed signals that do not correspond to a sequencing event in order to obtain higher quality sequencing information. The instant invention provides for such methods and systems.